Anopheles superpictus Grassi is one of the most widespread malaria vectors in Iran. Two morphologically independent forms of this mosquito, both at larval and adult stage as well as a great diversity in its mtDNA loci have been previously described in Iran. Because of existence of mtDNA haplotypes, co-occurrence of two forms in diverse climates as well as different roles of populations played in malaria transmission, we hypothesized the possibility of emerging species (or sub-species) within the taxon. We surveyed the molecular variation in sympatric and allopatric populations of the two forms, using sequences from the ribosomal-DNA spacer region (ITS2). This analysis revealed a high degree of polymorphism (32.3%) as well as a length polymorphism (357 vs. 378 bp) in the ITS2 region among the populations but not so among morphological forms. Further examination identified three different ITS2 sequences, designated as genotypes X, Y, and Z within species. Interestingly, while the sympatric Y and Z genotypes appear to be exclusive to the populations from the southeastern part of the country, genotype X is geographically separated and present in the North, the West, the South and the Central territories. The degree of divergence in ITS2 is much more than an intra-specific variation seen within the anopheline mosquitoes, and it points out the possibility of cryptic species within the taxon. Further studies are necessary to identify the species composition of the An. superpictus and their role played in the transmission of malaria in its geographical range.